The main goal of this application is the design, production and evaluation of a conceptually novel vaccine against Chikungunya virus (CHIKV). We propose a novel technology of "infectious" DNA (i-DNA) as CHIKV vaccine. A unique feature of this technology is that live attenuated CHIKV vaccine is launched in vivo from the i-DNA plasmid. In the i-DNA, the full-length copy of RNA genome of modified live attenuated IND vaccine 181/25 is placed in the plasmid in the context of optimized eukaryotic promoter and regulatory sequences. Transcription of the genomic viral RNA in vivo results in limited replication of attenuated virus in the tissues of vaccine recipient and induction of a protective immune response. Since the i-DNA represents a molecular clone, it will generate a uniform population of attenuated virus thus potentially improving safety. We will also prepare two i-DNA variants by de-optimization of translational codons within C-E2-E1 genes with the view to enhance genetic stability and vaccine safety. Experimental CHIKV i-DNA vaccines will be evaluated in vitro and in vivo along with the current 181/25 live attenuated vaccine. The vaccine antigens will be probed with a panel of human antisera from recent clinical CHIKV isolates in partnership with Sri Ramachandra University, Chennai, India. Immunogenicity and safety profiles will be determined in the young and aged mice as well as in immunosuppressed hamsters. Thus, characteristics of candidate i-DNA vaccines will be evaluated in the preclinical models of various age and immune status, which mimics human population and will provide accurate determination of safety and immunogenicity profiles of the vaccines. In summary, i-DNA vaccination will combine the simplicity of DNA vaccines with the exceptional efficacy of live attenuated vaccines. The i-DNA can potentially improve safety, does not require cold chain and is easy to manufacture and scale-up in emergency scenarios. Further, bacterially generated i-DNA will contain CpG motifs, which are expected to activate innate immune responses and improve immunogenicity. If successful, this technology may represent a revolutionary solution for vaccination against Chikungunya fever. PUBLIC HEALTH RELEVANCE: Chikungunya virus (CHIKV) is an emerging pathogen and a public health concern worldwide. The focus of this study is the design, production and evaluation of a conceptually novel vaccine against CHIKV. We hypothesize that safety and immunogenicity of live attenuated CHIKV vaccine can be improved by using the "infectious" DNA technology (i-DNA). This will result in a unique CHIKV vaccine, which will combine the simplicity of DNA vaccines with the exceptional efficacy of live attenuated vaccine. Such vaccine can be rapidly deployed during outbreaks and prevent spread of infection to non-affected areas.